Diptheria toxin is a procaryotic protein (molecular weight 63,000) that enters the cysol of most mammalian cells and arrests protein synthesis. The mechanism by which the protein is transported from a receptor at the exterior surface of the cell and past a membrane barrier to the cytosol is unknown. The objective of this proposal is to investigate the transport mechanism with the techniques of biochemistry and somatic cell genetics. There are four specific aims in the proposal. We have new evidence suggesting that the endocytic-lysosomal apparatus of the cell is involved in the transport process. The first aim is to further evaluate this suggestion. Second, the efficiency with which a cell transports the toxin to its cytosol is unknown and we will measure this in a mutant cell whose ability to transport the toxin is uncoupled from the cytotoxic effects of the toxin. Third, we will reconstitute the transport system in cells that lack this activity by fusing to them membranes from transport normal cells. Fourth, we will isolate and characterize mutants that are resistant to the lethal effects of the toxin because they are transport-deficient. These mutants will provide additional systems for studying the transport mechanism and how it participates in the normal functions of the cell.